The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay (original) (raw)
Figure 2
MAGOH-Y14 are recruited to the EJC by eIF4A3.
(A) Scheme of the positions of the MAGOH mutants in the EJC. The interactions with UPF3b and PYM are indicated by arrows. BTZ residues present in the crystal structure are displayed in red, an arbitrary shape of full length BTZ is suggested in light red. The EJC structure was rendered using PyMOL [54] with structural data deposited in the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do; ID: 2j0s). (B) Immunoprecipitations were done from RNAse A-treated lysates of HeLa cells that were transfected with FLAG-MAGOH and FLAG-MAGOH mutants or unfused FLAG as negative control together with V5-tagged BTZ, UPF3b, eIF4A3, PYM, and Y14. Co-precipitated proteins were detected by immunoblotting using an anti V5 antibody. (C) Northern blot analysis of RNA from HeLa cells that were transfected with expression plasmids for λNV5-tagged MAGOH or mutants of MAGOH together with the 4boxB reporter plasmid and the transfection control plasmid. Percentages (%) represent the mean of four independent experiments±standard deviations (SD). Bottom panel: expression levels of the tethered MAGOH mutant proteins were detected by immunoblot analysis with a V5-specific antibody. GFP served as internal loading control. (D) Splicing reactions using MINX as substrate RNA were supplemented with extracts expressing the indicated FLAG-tagged MAGOH mutants or unfused FLAG-tag as negative control. Reactions were immunoprecipitated with FLAG affinity gel. Twelve percent of the total input material was loaded in the input panel. (E) Splicing reactions and immunoprecipitations were performed as in (D) with the mutated MINX GG transcript.